NKT cells promote Th1 immune bias to dengue virus that governs long-term protective antibody dynamics

NKT cells are innate-like T cells, recruited to the skin during viral infection, yet their contributions to long-term immune memory to viruses are unclear. We identified granzyme K, a product made by cytotoxic cells including NKT cells, as linked to induction of Th1-associated antibodies during primary dengue virus (DENV) infection in humans. We examined the role of NKT cells in vivo using DENV-infected mice lacking CD1d-dependent (CD1ddep) NKT cells. In CD1d-KO mice, Th1-polarized immunity and infection resolution were impaired, which was dependent on intrinsic NKT cell production of IFN-γ, since it was restored by adoptive transfer of WT but not IFN-γ–KO NKT cells. Furthermore, NKT cell deficiency triggered immune bias, resulting in higher levels of Th2-associated IgG1 than Th1-associated IgG2a, which failed to protect against a homologous DENV rechallenge and promoted antibody-dependent enhanced disease during secondary heterologous infections. Similarly, Th2 immunity, typified by a higher IgG4/IgG3 ratio, was associated with worsened human disease severity during secondary infections. Thus, CD1ddep NKT cells establish Th1 polarity during the early innate response to DENV, which promotes infection resolution, memory formation, and long-term protection from secondary homologous and heterologous infections in mice, with consistent associations observed in humans. These observations illustrate how early innate immune responses during primary infections can influence secondary infection outcomes.

For quantification of cytokines, cDNA was synthesized from the RNA isolated from tissues with the iScript Select cDNA Synthesis Kit (Bio-Rad) to amplify RNA containing a poly(A) tail.Real-time PCR was performed with SYBR Green reagent using Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad; Hercules, CA).The results were averaged from triplicates from multiple (n=3-5 mice) FPs and dLNs, and the gene expression of types-I, -II, or NKT-like cells from uninfected or DENV2-infected WT mice were calculated relative to the total NKT cells isolated from uninfected WT mice.

TCR Sequencing
WT or CD1d-KO mice, n=3 per group, were injected I.P. with PBS or DENV2 at 10 6 PFU.Three days post infection, mesenteric LNs and Iliac LNs were collected and pooled.LNs were made into single cell suspensions.Live/dead staining was performed with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (Invitrogen™, #L34975), prior to staining with PerCP-Cy5.5-conjugatedanti-mouse antibody and PEconjugated anti-mouse NK1.1.Live CD3 + NK1.1 + cells were then sorted (50,000 cells per group).Sorted cells were stored in RNAprotect cell Reagent (Qiagen, #76526) and sent to iRepertoire (Huntsville, AL) for sequencing of the TCR-Vb region.Data were analyzed using iRepetoire's software.For the diversity index, they define diversity as 100 minus the area under the curve between the percentage of total reads and the percentage of unique CDR3s, when unique CDR3s are sorted by frequency from largest to smallest.

NKT cell purification and adoptive transfer
Spleen and lymph nodes (Iliac, mesenteric and popliteal) were harvested from wildtype (WT) or IFNg-KO 6 weeks old mice.The organs were incubated in collagenase at 37°C for 30 min and prepared as single cell suspensions using 70µm cell strainers (Falcon).NKT cells were isolated from the single cell suspensions using the NK1.1 + iNKT cell isolation kit mouse (Miltenyibiotec, #130-096-513) according to the manufacturer recommendations.After purification, NKT cells were transferred into 6 week old CD1d-KO mice, by injecting 5x10 5 cells per mouse, IP.One group of CD1d-KO mice received purified NKT cells from WT mice and another group of CD1d-KO mice received NKT cells purified from IFNg-KO mice.Twenty-four hours post-transfer, both groups were infected by FP injection with 1x10 5 PFU of DENV2, strain Eden2.Three days post-infection, FPs and popliteal dLNs were harvested for flow cytometry or qPCR analysis.

Quantification of antibody titers
ELISAs were performed following previously published protocols (5,6) with minor modifications.In brief, black 96-well half-area microtiter plates (Costar) were coated with 1x10 5 PFU/mL of DENV2, Eden2 strain, in carbonate buffer (15mM Na2CO3, 35mM NaHCO3, pH 9.6) overnight at 4°C.Plates were blocked with 3% non-fat dry milk powder in PBS 0.05% Tween-20 for 2 hours at room temperature.Plates were washed and 2-fold serial dilutions, beginning at 1:128, of mouse sera in complete sample diluent (PBS, BSA 1%, nonfat dry milk 1% W/V, normal goat serum 5% V/V (Sigma-Aldrich), and tween-20 0.05% V/V) were added, and incubated 2 hours at room temperature.After washing, alkaline phosphatase-conjugated anti-mouse IgG1 or IgG2a detection antibody (BD Biosciences, #557272 and #553389, respectively) were added at a 1:8000 dilution in secondary antibody diluent (PBS, BSA 0.5%, normal goat serum 5% V/V (Sigma-Aldrich), and tween 20 0.05% V/V) and incubated for 2 hours at room temperature.After washing, plates were incubated with Attophos® AP fluorescent substrate system (Promega) for 45 min at room temperature in the dark and then read at 440nm (excitation)/560nm (emission) using a plate reader (Spark 10M, Tecan).Serum dilutions were considered positive if the relative light units (RLUs) measured were at least two-fold higher than the value of the naïve serum at the same dilution.Similarly, for the total specific anti-DENV2 IgG titers, the serial dilution started at 1:64.The secondary antibody, goat anti-mouse IgG-AP (Southern Biotech, #10130-04), was used at a dilution of 1:10000.

ADE assays
In vivo ADE assays were performed using homologous and heterologous challenges.For the heterologous challenge, WT mice or CD1d-KO mice were infected by IP with 10 6 PFU of DENV2, Eden2 strain.After 4 weeks, the sera were collected and IgG was purified with NAb™ Protein A/G Spin Kit (ThermoScientic).IgG (1µg) from DENV2-immune WT or DENV2-immune CD1d-KO or from naive mice was incubated with with 2x10 5 PFU of DENV1, Eden1 strain, for 1 hour at 37°C.For homologous challenge, mice were infected and IgGs were purified as above.IgG (10µg) from DENV2-immune WT, DENV2-immune CD1d-KO or from naive mice was incubated with with 1x10 5 PFU of DENV2, Eden2 strain, for 1 hour at 37°C.After this incubation to generate immune complexes, they were injected by FP to IFNαR/IFNγR mice (n=5 mice per group).Five days post infection, FP skin and dLN were haversted for RNA extraction with the RNaeasy Kit (Qiagen) and one step qPCR were performed with SuperScript™ III Platinium™ One-step qRT-PCR System (Invitrogen), used with either DENV2 or DENV1-specific primers and probes, as appropriate given the homologous or heterologous challenge, to quantify the viral load.
For the survival experiment, 1µg of IgG from DENV2-immune WT or DENV2-immune CD1d-KO mice were incubated with with 2x10 6 PFU of DENV1, Eden1 strain, for 1 hour at 37°C.Mice were injected I.P. and monitored every day for duration of the experement.Humane endpoints including weight loss were used as surrogates for survival.

Human dengue biomarker assays
Serum samples from pediatric dengue patients (<12 years of age) that were recruited as previously described (7) were used for biomarker assays.In brief, confirmed dengue patients were prospectively recruited at Lady Ridgeway Hospital, Colombo, Sri Lanka upon meeting the following study criteria: having a fever of ≥ 38°C for less than 72h, hospital admission, and confirmation of DENV infection by NS1 antigen test at the time of recruitment.Longitudinal samples obtained 1-3 days, 4-5 days, and/or 6-7 days post fever onset were used as indicated in figure legends.The final DF versus DHF diagnoses were according to the 1997 WHO critera, and viral RNA quantification was performed as previously reported (7).Research was designed to be compliant with the Declaration of Helsinki.Ethical approvals were obtained from the Institutional Review Boards of Lee Kong Chian School of Medicine, Nanyang Technological University (NTU), Singapore, National University of Singapore and the Ethics Review Committee of Faculty of Medicine, University of Colombo, Sri Lanka.Written informed consent was given by a parent or guardian of the patient.
Samples were selected for biomarkers testing on the basis of having sufficient remaining volume following their use for DENV confirmation and prior research objectives to complete the assays as well as on the basis of having matched longitudinal samples for the same patients.Primary versus secondary infections were determined by Panbio Dengue IgG Capture ELISA (Catalog number: 01PE10) and Panbio Dengue IgM Capture ELISA (Catalog number: 01PE20), both from PanBio Diagnostics.Primary cases were identified as IgM+IgG− and secondary cases were identified as IgM+IgG+ (8).ELISAs and biomarker assays were performed by a blinded study team member.

Software
Prism 5 and Excel were used to determine statistical significance.Heat map in Figure 8A was made with Heatmapper software (9).Violin plots in Figures 8B-D were made with PlotsofData (10).All diagrams were made with Biorender.com.

Supplementary Figure 2 :
Unaltered cellular parameters between WT and CD1d-KO mice following DENV infection.Quantification of NK cells in the (A) dLN and (B) FP skin and (C) NKT cells in the dLNs of WT and CD1d-KO mice following DENV infection.(D) Quantification of CD1d ind NKT cell subsets in the dLNs, in parallel to Figure 2F, showed no significant difference between WT and CD1d-KO mice following DENV infection.(E) Immunostaining of WT mice at day 3 post-infection confirmed the presence of NKT cells in the skin (image representative of n=3 mice).(F) Low and (G) High magnification confocal images of dLNs immunostained for NK1.1 and CD3, showing NKT (NK1.1 + CD3 + ) (Images representative of n=3 per group).